This prject is designed to extend studies regarding the oligonucleotide recognition sequence(s) and the ribosomal RNA (rRNA) conformational requirements involved in the mechanism of site selection by the rRNA methylases and to elucidate how the binding of ribosmal proteins to precursor rRNA (p-rRNA) facilitates the formation of these methylation sites during specific stages of ribosomal assembly. Two possibilities for site-formation will be explored: (a) the secondary and tertiary conformation of p-rRNA does not provide the necessary single-strandedness at the recognition site until its conformation is altered during the assembly process. (b) the potential site exists but is initially masked by nonspecifically bond proteins until the specific binding of r-proteins exposes the sites. Initially, these studies will emphasize one r-methylase, namely rRNA-adenine (N6 minus) methylase of E. coli and will encompass (a) in vitro metylation of homologous p-rRNA t determine the umber and identification of the methylation sites, (b) isolation of unique methylase species responsible for the methylation of each unique site, (c) isolation of mutants deficient in rRNA-adenine (N6 minus) methylase, (d) examination of ribosomal precursor-particles to determine the extent of rRNA modification and protein binding required before 6-methyladenine synthesis occurs, (e) in vivo methylation of p-rRNA synthesized in the presence of chloramphenicol, (f) inhibition of methylases by r-prteins, (g) in vitro methylation of the p-rRNA in normal ribosomal precursor-particles and partially reconstituted particles, and (h) a study to determin whether the association of the methylase with ribosomal fractions represents a selective binding to pecific structures in the ribosomal assembly process.